In most eukaryotic cells (including yeast and humans), the iron insertion step for synthesis of heme occurs exclusively within mitochondria. Likewise, iron insertion for formation of new FeS clusters can occur within mitochondria. Since the mitochondrial inner membrane is impermeable to ions (a requirement for maintaining the electrogenic membrane potential), the question arises of how iron for heme and FeS clusters gets into mitochondria. Recent data from several labs have demonstrated involvement of mitochondrial carrier proteins Mrs3 and Mrs4 in yeast, and orthologs mitoferrin 1 and 2 in vertebrates. However, many aspects of the iron transport process remain unknown. Frataxin a small conserved mitochondrial protein is involved downstream in iron use for heme and FeS clusters within mitochondria, but its precise function is also unclear. Here we propose three genetic and biochemical aims to delve further into this process. Aim 1) will seek to characterize the iron transport process in isolated yeast mitochondria in short time frames. The role of Mrs3 and Mrs4, and the role of the electrogenic membrane potential in mediating iron transport will be studied. Targeted mutations in the transporters, specifically in the hypothetical substrate binding site, will be evaluated, and the role of a giant protein complex of roughly 660 kDa containing the transporter(s) will be investigated. Aim 2) will ascertain the role of frataxin in FeS and heme synthesis within mitochondria. The amount of frataxin in mitochondria will be varied over a large range and targeted mutations will be analyzed specifically mutations in interaction sites with iron, with Isu1 (scaffold for FeS cluster assembly) and with ferrochelatase (enzyme for heme synthesis). Assays in isolated mitochondria in short time frames will allow direct effects to be distinguished from secondary effects. In aim 3), novel genes and proteins connected to iron transport to mitochondria will be sought by means of an ongoing screen to identify mutations that are synthetically lethal with mrs3 and mrs4. Early results show that single mutants (e.g. dre2, tsa1) identified in this screen are involved in iron metabolism. Iron transport into mitochondria is an essential conserved process in eukaryotes, required for heme synthesis and FeS cluster assembly. Proposed studies will be performed in yeast mitochondria, but the organization of mitochondria is highly conserved between yeast and humans. The particular yeast proteins involved in iron transport and use in mitochondria have human orthologs (frataxin, mitochondrial carriers). Defects in these proteins have been implicated in porphyria, anemia and neurodegeneration, and so results obtained in with yeast will be relevant to human disease.